Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.

荧光定位显微镜已经实现了近分子分辨率，能够揭示超结构，具有广泛的应用，尤其是在细胞生物学中。然而，在三维上以及在第一细胞层之外的生物组织内部实现这种分辨率仍然具有挑战性。在这里，我们介绍SELFI，这是在多细胞标本和组织中进行3D单分子定位的框架。该方法依靠显微镜的点扩散函数（PSF）内产生的自干扰来同时编码等相和强度荧光信号，这些信号共同提供了发射器的3D位置。我们将SELFI与常规定位显微镜相结合，以可视化F-actin 3D细丝网络，并揭示了人类诱导的多能干细胞中转录因子OCT4在未清除的组织球体内部深度达50 µm的空间分布。SELFI为完整组织中天然细胞过程的纳米级研究铺平了道路。